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To date, a completely in vitro repopulated tissue-engineered heart valve has not been developed. This study focused on sequentially seeding 2 cell populations onto porcine decellularized heart valve leaflets (HVL) and pericardia (PER) to obtain fully repopulated tissues. For repopulation of the interstitium, porcine valvular interstitial cells (VIC) and bone marrow-derived mesenchymal stem cells (BM-MSC) or adipose tissue-derived stem cells (ADSC) were used. In parallel, the culture medium was supplemented with ascorbic acid 2-phosphate (AA) and its effect on recolonization was investigated. Subsequently and in order to obtain an endothelial surface layer similar to those in native HVL, valvular endothelial cells (VEC) were seeded onto the scaffolds. It was shown that VIC efficiently recolonized HVL and partially also PER. On the other hand, stem cells only demonstrated limited or no subsurface cell infiltration of HVL and PER. Interestingly, the addition of AA increased the migratory capacity of both stem cell populations. However, this was more pronounced for BM-MSC, and recolonization of HVL appeared to be more efficient than that of PER tissue. VEC were demonstrated to generate a new endothelial layer on HVL and PER. However, scanning microscopy revealed that these endothelial cells were not allowed to fully spread onto PER. This study provided a proof of concept for the future generation of a bioactive tissue-engineered heart valve by showing that bioactive HVL could be generated in vitro within 14 days via complete repopulation of the interstitium with BM-MSC or VIC and subsequent generation of an entirely new endothelium.

Fat grafting has become a widespread technique for different reconstructive and esthetic purposes. However, the disadvantage of fat grafting is the unpredictable resorption rate that often necessitates repetitive procedures, which in turn may have an impact on the morbidity. During the immediate, post-graft, ischemic period, cells survive due to the process of plasmatic imbibition. This biological phenomenon precedes the ingrowth of neo-capillaries that eventually nourish the graft and help establish a long-term homeostatic equilibrium. Both partners, the graft and the recipient bed, contribute to the revascularization process. Hypothetically, enrichment of the recipient site with autologous plasma could have a beneficial role to enhance fat graft survival. We investigated whether plasma supported the viability of the lipoaspirate (LA) material. Plasma was isolated from blood samples collected from eight patients during the elective lipofilling procedures. An in vitro study assessed the viability of LA cells using plasma as a culture medium compared to the traditional culture media. In vitro analysis confirmed sustained viability of LA cells compared to the standard media and control media during 7 consecutive days. The behavior of the fat grafts in plasma showed similarities with those incubated in the traditional culture media. In future, these findings could be translated to a clinical setting. Plasma is the only autologous substrate available in large quantities in the human body. The addition of the supporting agents, such as plasma, could contribute to a better graft survival with more stable clinical outcomes in the long term. The rationale behind the technique is based on the phenomenon of plasmatic imbibition and the reasoning that the extracellular matrix plays a pivotal role in cellular survival.

To date an optimal decellularization protocol of heart valve leaflets (HVL) and pericardia (PER) with an adequate preservation of the extracellular matrix (ECM) is still lacking. This study compares a 4 day Triton X-100-based protocol with faster SDC-based protocols for the decellularization of cardiac tissues. Decellularized and non-treated HVL and PER were processed for histological, biochemical and mechanical analysis to determine the effect of these agents on the structure, ECM components, and biomechanical properties. Tissues treated with SDC-based protocols still showed nuclear material, whereas tissues treated with Triton X-100 1% + ENZ ± TRYP were completely cell free. For both decellularized tissues, an almost complete washout of glycosaminoglycans, a reduction of soluble collagen and an alteration of the surface ultrastructure was observed. Interestingly, only the elastic fibers of pericardial tissue were affected and this tissue had a decreased maximum load. This study showed that both detergents had a similar impact on the ECM. However, Triton X-100 1% +DNase/RNase (ENZ) ± Trypsin (TRYP) is the only protocol that generated completely cell free bioscaffolds. Also, our study clearly demonstrated that the decellularization agents have more impact on pericardial tissues than on heart valve leaflets. Thus, for the purpose of tissue engineering of heart valves, it is advisable to use valvular rather than pericardial matrices.

In meniscus tissue engineering strategies, enhancing the matrix quality of the neomeniscal tissue is important. When the differentiated phenotype of fibrochondrocytes is lost, the quality of the matrix becomes compromised. The objective of this study was to produce uniform fibrochondrocyte micro-aggregates with desirable phenotype and tissue homogeneity in large quantities using a simple and reproducible method. Furthermore, we investigated if hypoxia could enhance the matrix quality. Porcine fibrochondrocytes were expanded at 21% oxygen until passage 3 (P3) and a gene expression profile was determined. P3 fibrochondrocytes were cultivated in chondrogenic medium at 5 and 21% oxygen in high-throughput agarose chips containing 2,865 microwells 200 µm in diameter. Evaluation included live/dead staining, histological examination, immunohistochemistry, dimethylmethylene blue assay and real-time reverse transcriptase quantitative polymerase chain reaction of the micro-aggregates. Gene expression analysis showed a drastic decline in collagen II and high expression of collagen I during monolayer culture. After 4 days, uniform and stable micro-aggregates could be produced. The redifferentiation and matrix quality of the hypoxic cultured micro-aggregates were enhanced relative to the normoxic cultures. Sulfated glycosaminoglycan synthesis was significantly higher, and collagen II expression and the collagen II/collagen I ratio were significantly upregulated in the hypoxic cultures. High-throughput production of uniform microtissues holds promise for the generation of larger-scale tissue engineering constructs or optimization of redifferentiation mechanisms for clinical applications.

Allylamine was plasma, post–irradiation grafted and UV polymerized onto UHMWPE surface to improve its cell adhesion and proliferation properties as well as to increase its wettability. For plasma polymerization, samples were coated using different process parameters. The effect of the various processes and process parameters on the physical and chemical properties of the deposited allylamine coatings was evaluated by different physical and chemical characterization techniques. Wettability, surface chemistry, thickness and surface morphology were determined. Stability and aging tests for a 7 day period were also investigated. Moreover, cell tests using human foreskin fibroblasts were performed and evaluations were conducted after 1 and 7 days of culturing. Different chemistries and surface morphologies were induced after each treatment and various nitrogen and oxygen functionalities were incorporated making the surface more hydrophilic. Coatings were relatively stable and cell viability was found to be significantly higher on plasma and UV treated UHMWPE compared to plain UHMWPE; this was confirmed by the overall cell morphology.

Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 trans men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

Hydrogel biomaterials can be easily enriched with bioactive substances such as the mineralization-promoting enzyme alkaline phosphatase (ALP). In this study, poly(vinyl alcohol) (PVA) hydrogels designed for osteochondral regeneration containing incorporated ALP were mineralized with calcium phosphate (CaP) and magnesium phosphate (MgP) by incubation in solutions of 0.1 M calcium or magnesium glycerophosphate (CaGP, MgGP). Hydrogels incubated in water served as controls. More mineral was formed in hydrogels incubated in CaGP than in MgGP. Rheometry revealed that mechanical strength (storage modulus) decreased in the order: CaGP>MgGP>water. Physicochemical charaterization showed that hydrogels incubated in CaGP appeared to be mineralized with apatite and amorphous CaP, while hydrogels incubated in MgGP appeared to be mineralized with plate-like MgP crystals and amorphous MgP. Hydrogels incubated in water were devoid of mineralization. Cell viability testing showed that proliferation on hydrogels incubated in MgGP was comparable to that on non-mineralized samples and superior to that on hydrogels incubated in CaGP. The results prove the principle of enzymatic mineralization of PVA hydrogels with CaP and MgP. Further work may concentrate on in vivo evaluation of the suitability of these mineralized hydrogels for bone or osteochondral regeneration applications.

The present study focuses on the alkaline phosphatase (ALP) mediated formation of apatitic minerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnated with different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatite while the structures differ as a function of the ALP concentration. Cellular adhesion, proliferation, and colonization of osteogenic MC3T3 cells improve on the mineralized SFP scaffolds. These findings suggest a simple process to generate mineralized scaffolds that can be used to enhanced bone tissue engineering-related utility.

The present study investigates the effect of galactosylated gelatin on encapsulated HepG2 cells. Methacrylamide modified gelatin is evaluated and compared with its galactosylated counterpart with respect to effects on viability, morphological characteristics, proliferation, and the expression of hepatocyte specific markers. The research reveals that further modifications of methacrylamide modified gelatin are possible without affecting the survival of the encapsulated cells (viability of 90%). Moreover, the study demonstrates a clear and long-term (up to 21 d) improvement in hepatocyte specific gene expression when the cells are encapsulated in the galactosylated gelatin. It is concluded that the use of galactosylated gelatin derivates supports the hepatocyte phenotype.

In this work, PCL surfaces are exposed to a medium pressure DBD operating in dry air, argon and helium to improve PCL-cell interactions. Two PCL sample types are examined in detail: flat PCL samples and 3D porous PCL scaffolds. From XPS measurements, it is found that plasma treatment results in the incorporation of oxygen and nitrogen on the PCL surface. A higher oxygen content has also been detected inside the porous PCL structure, indicating that the plasma is capable of penetrating this structure without losing its modifying ability. To investigate the plasma treatment effect on PCL-cell interactions, cell culture tests have also been performed. Cell culture tests on flat PCL samples indicate that the applied plasma treatment increases cell numbers and improves cell attachment, independent of the used discharge gas. Moreover, a higher cell amount is detected on plasma-treated PCL scaffolds and proves that cells also grow better on plasma-treated scaffolds, suggesting the positive influence of plasma treatment on cell adhesion and proliferation. These cell culture results thus clearly prove the beneficial effect of plasma treatment on PCL-cell interactions.

For most tissue engineering applications, surface modification and sterilization of polymers are critical aspects determining the implant success. The first part of this study is thus dedicated to modifying polycaprolactone (PCL) surfaces via plasma treatment using a medium pressure dielectric barrier discharge, while the second part focuses on the sterilization of plasma-modified PCL. Chemical and physical surface changes are examined making use of water contact angle goniometry (WCA), x-ray photoelectron spectroscopy and atomic force microscopy. Bioresponsive properties are evaluated by performing cell culture tests. The results show that air and argon plasmas decrease the WCA significantly due to the incorporation of oxygen-containing functionalities onto the PCL surface, without modifying its morphology. Extended treatment times lead to PCL degradation, especially in the case of air plasma. In addition to surface modification, the plasma potential to sterilize PCL is studied with appropriate treatment times, but sterility has not been achieved so far. Therefore, plasma-modified films are subjected to UV, H2O2 plasma (HP) and ethylene oxide (EtO) sterilizations. UV exposure of 3 h does not alter the PCL physico-chemical properties. A decreased wettability is observed after EtO sterilization, attributable to the modification of PCL chain ends reacting with EtO molecules. HP sterilization increases the WCA of the plasma-treated samples, presumably due to the scission of the hydrophilic bonds generated during the prior plasma treatments. Moreover, HP modifies the PCL surface morphology. For all the sterilizations, an improved cell adhesion and proliferation is observed on plasma-treated films compared to untreated ones. EtO shows the lowest proliferation rate compared to HP and UV. Overall, of the three sterilizations, UV is the most effective, since the physical alterations provoked by HP might interfere with the structural integrity when it comes to 3D scaffolds, and the chemical modifications caused by EtO, in addition to its toxicity, interfere with PCL bioactivity.

In this work, medium pressure plasma treatment of polylactic acid (PLA) is investigated. PLA is a biocompatible aliphatic polymer, which can be used for bone fixation devices and tissue engineering scaffolds. Due to inadequate surface properties, cell adhesion and proliferation are far less than optimal and a surface modification is required for most biomedical applications. By using a dielectric barrier discharge (DBD) operating at medium pressure in different atmospheres, the surface properties of a PLA foil are modified. After plasma treatment, water contact angle measurements showed an increased hydrophilic character of the foil surface. X-ray photoelectron spectroscopy (XPS) revealed an increased oxygen content. Cell culture tests showed that plasma modification of PLA films increased the initial cell attachment both quantitatively and qualitatively. After 1 day, cells on plasma-treated PLA showed a superior cell morphology in comparison with unmodified PLA samples. However, after 7 days of culture, no significant differences were observed between untreated and plasma-modified PLA samples. While plasma treatment improves the initial cell attachment, it does not seem to influence cell proliferation. It has also been observed that the difference between the 3 discharge gases is negligible when looking at the improved cell-material interactions. From economical point of view, plasma treatments in air are thus the best choice.

Interest is growing in the use of hydrogels as bone tissue-engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium-crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite-like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP-OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP-mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA.

Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering and cell therapy. The large-scale banking of hESCs for research and future clinical application requires economic, defined, and xeno-free cryopreservation protocols. In this study, the possibility to substitute knockout serum replacement (KO-SR) in the cryopreservation process with vegetal and synthetic hydrolysates was investigated. To our knowledge, the use of hydrolysates in hESC cryopreservation has not been yet explored. Initially, 3 different hydrolysates (Ultrapep Soy, Hypep 4601 and EX-CELL(®) CD Hydrolysate Fusion) were tested in the cryopreservation solution. A concentration of 8 mg/mL EX-CELL CD Hydrolysate Fusion in the cryopreservation solution leads to the highest recovery ratio; thus, this solution was selected for additional cryopreservation experiments. To ensure reproducibility of the results, 3 hESC lines (HS181, H9, and BG01) were used. The hESCs were collected prefreezing by application of collagenase IV and cell dissociation solution. Experiments showed that it was feasible to substitute the KO-SR in both the cryopreservation solution as the thawing solution. The obtained recovery ratios were comparable to those obtained with KO-SR (no statistical significant difference; Student's t-test, P0.05). Further optimization protocols showed a doubling of the obtained recovery ratio after addition of Rock-inhibitor Y-27632 post-thawing. The expansion profile and pluripotency analysis revealed no changes in normal hESC behavior. We conclude that the application of vegetal or synthetic hydrolysates is suitable for xeno-free hESC cryopreservation.

An electronic sensor system for urinary bladder pressure monitoring requires an imbedding into a biocompatible, flexible, and liquid-impermeable material. Poly(dimethylsiloxane) (PDMS) was selected in the present set-up as packaging material because it fulfills the abovementioned requirements. However, the surface of PDMS is hydrophobic and causes undesired interactions with salts, proteins, and cells present in urine. To reduce possible interactions of urine salts in the urinary bladder, monomers, [2-(methacryloyloxy)ethyl]-dimethyl-3-sulfopropyl-ammonium hydroxide (sulfobetaine) and 2-acrylamido-2-methylpropyl sulfonic acid, were grafted onto the surface through oxygen plasma treatment. A reduction in salt deposition between the pure PDMS and the modified PDMS was observed both in vitro (artificial urine flow over the surface) and in vivo (implants into the urinary bladder of experimental pigs). Additionally, a 10-fold reduction in salt deposition was observed in vitro due to grafting of the monomers onto the surface. These modified PDMS materials proved also to be biocompatible in cell cultures, which was further confirmed by histological screening of the bladder tissue after implantation in an in vivo pig model.

Pluronic® F127 is a biocompatible, injectable, and thermoresponsive polymer with promising biomedical applications. In this study, a chemically modified form, i.e., Pluronic ALA-L with tailored degradation rate, was tested as an encapsulation vehicle for osteoblastic cells. UV cross-linking of the modified polymer results in a stable hydrogel with a slower degradation rate. Toxicological screening showed no adverse effects of the modified Pluronic ALA-L on the cell viability. Moreover, high viability of embedded cells in the cross-linked Pluronic ALA-L was observed with life/death fluorescent staining during a 7-day-culture period. Cells were also cultured on macroporous, cross-linked gelatin microbeads, called CultiSpher-S® carriers, and encapsulated into the modified cross-linked hydrogel. Also, in this situation, good cell proliferation and migration could be observed in vitro. Preliminary in vivo tests have shown the formation of new bone starting from the injected pre-loaded CultiSpher-S® carriers.

In the last decade, substantial research in the field of post-plasma grafting surface modification has focussed on the introduction of carboxylic acids on surfaces by grafting acrylic acid (AAc). In the present work, we report on an alternative approach for biomaterial surface functionalisation. Thin poly-ε-caprolactone (PCL) films were subjected to a dielectric barrier discharge Ar-plasma followed by the grafting of 2-aminoethyl methacrylate (AEMA) under UV-irradiation. X-ray photoelectron spectroscopy (XPS) confirmed the presence of nitrogen. The ninhydrin assay demonstrated, both quantitatively and qualitatively, the presence of free amines on the surface. Confocal fluorescence microscopy (CFM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise the grafted surfaces, indicating the presence of pAEMA. Static contact angle (SCA) measurements indicated a permanent increase in hydrophilicity. Furthermore, the AEMA grafted surfaces were applied for comparing the physisorption and covalent immobilisation of gelatin. CFM demonstrated that only the covalent immobilisation lead to a complete coverage of the surface. Those gelatin-coated surfaces obtained were further coated using fibronectin. Osteosarcoma cells demonstrated better cell-adhesion and cell-viability on the modified surfaces, compared to the pure PCL films.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation. Stem Cells 2015;33:2686–2698

The present work describes for the first time the production of self-supporting low gelatin density (

The present work describes the development and the evaluation of cryogel-poly-epsilon-caprolactone combinatory scaffolds for bone tissue engineering. Gelatin was selected as cell-interactive biopolymer to enable the adhesion and the proliferation of mouse calvaria pre-osteoblasts while poly-epsilon-caprolactone was applied for its mechanical strength required for the envisaged application. In order to realize suitable osteoblast carriers, methacrylamide-functionalized gelatin was introduced into 3D printed poly-epsilon-caprolactone scaffolds created using the Bioplotter technology, followed by performing a cryogenic treatment which was concomitant with the redox-initiated, covalent crosslinking of the gelatin derivative (i.e. cryogelation). In a first part, the efficiency of the cryogelation process was determined using gel fraction experiments and by correlating the results with conventional hydrogel formation at room temperature. Next, the optimal cryogelation parameters were fed into the combinatory approach and the scaffolds developed were characterized for their structural and mechanical properties using scanning electron microscopy, micro-computed tomography and compression tests respectively. In a final part, in vitro biocompatibility assays indicated a good colonization of the pre-osteoblasts and the attachment of viable cells onto the cryogenic network. However, the results also show that the cellular infiltration throughout the entire scaffold is suboptimal, which implies that the scaffold design should be optimized by reducing the cryogel density.

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Item does not contain fulltext Hydrogels of biocompatible calcium-crosslinkable polysaccharide gellan gum (GG) were enriched with bioglass particles to enhance (i) mineralization with calcium phosphate (CaP); (ii) antibacterial properties and (iii) growth of bone-forming cells for future bone regeneration applications. Three bioglasses were compared, namely one calcium-rich and one calcium-poor preparation both produced by a sol-gel technique (hereafter referred to as A2 and S2, respectively) and one preparation of composition close to that of the commonly used 45S5 type (hereafter referred to as NBG). Incubation in SBF for 7 d, 14 d and 21 d caused apatite formation in bioglass-containing but not in bioglass-free samples, as confirmed by FTIR, XRD, SEM, ICP-OES, and measurements of dry mass, i.e. mass attributable to polymer and mineral and not water. Mechanical testing revealed an increase in compressive modulus in samples containing S2 and NBG but not A2. Antibacterial testing using biofilm-forming meticillin-resistant staphylococcus aureus (MRSA) showed markedly higher antibacterial activity of samples containing A2 and S2 than samples containing NBG and bioglass-free samples. Cell biological characterization using rat mesenchymal stem cells (rMSCs) revealed a stimulatory effect of NBG on rMSC differentiation. The addition of bioglass thus promotes GG mineralizability and, depending on bioglass type, antibacterial properties and rMSC differentiation.

Autotransplantation of immature teeth can have a success rate of almost 98% if the tooth is atraumatically transplanted from the donor site to a suitable acceptor site and the extraoral time is kept to a minimum. When the tooth cannot be transplanted immediately, cryopreservation and storage in a tooth bank offer new possibilities for autotransplantation. However, the effect of cryopreservation on the revascularization of transplanted teeth is still unknown. The purpose of this study was to examine revascularization in immature teeth that have an open apex and in mature teeth that have had the apex cut. The study was carried out on 16 teeth in 2 dogs; 8 teeth were removed and immediately transplanted to the contralateral position and 8 teeth were cryopreserved and transplanted 1 week later. The results show that: (1) teeth can revascularize after autotransplantation if the original pulp tissue is removed at the time of extraction, (2) there is no significant difference in the amount of revascularization between teeth stored in a tooth bank for 7 days and those immediately transplanted without freezing, and (3) there is no difference in the ingrowth of new pulpal tissue between mature apicoectomized teeth and immature teeth. (Am J Orthod Dentofacial Orthop 2001;119:346-52)

The present study focuses on the alkaline phosphatase (ALP) mediated formation of apatitic minerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnated with different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatite while the structures differ as a function of the ALP concentration. Cellular adhesion, proliferation, and colonization of osteogenic MC3T3 cells improve on the mineralized SFP scaffolds. These findings suggest a simple process to generate mineralized scaffolds that can be used to enhanced bone tissue engineering-related utility.

The present study investigates the effect of galactosylated gelatin on encapsulated HepG2 cells. Methacrylamide modified gelatin is evaluated and compared with its galactosylated counterpart with respect to effects on viability, morphological characteristics, proliferation, and the expression of hepatocyte specific markers. The research reveals that further modifications of methacrylamide modified gelatin are possible without affecting the survival of the encapsulated cells (viability of 90%). Moreover, the study demonstrates a clear and long-term (up to 21 d) improvement in hepatocyte specific gene expression when the cells are encapsulated in the galactosylated gelatin. It is concluded that the use of galactosylated gelatin derivates supports the hepatocyte phenotype.

In this article, a medium-pressure DBD plasma treatment is used to improve the cell–material interaction of a polyhydroxybutyrate/polyhydroxyvalerate (PHB/PHV) film. PHB/PHV is a biodegradable natural polyester, used for different biomedical applications, including sutures, repair devices, and bone marrow scaffolds. The cell adhesion onto PHB/PHV is far less than optimal due to inadequate surface properties, and a surface modification is usually necessary to be able to use the full potential. Medium-pressure plasma treatments, in different atmospheres, are used to change the surface properties of a PHB/PHV foil. The hydrophilic character could be increased, as shown by water contact angle measurements. X-ray photoelectron spectroscopy (XPS) revealed an increased oxygen and nitrogen content. Cell culture test with human foreskin fibroblasts showed that the plasma was able to improve cell adhesion (both quantitatively and qualitatively). Both an increase in the number of adherent cells and an improved morphology were obtained after plasma treatment. After 7 days, a confluent cell layer could be observed on plasma-treated samples. The differences between the three discharge gases are negligible when looking at the improved cell–material interactions. From economical point of view, treatments in air are thus the best choice. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Summary form only given. Biomedical polymers have a great potential in medicine due to their biocompatible nature and versatility. However, their low surface energy leads to a cell adhesion and proliferation that is far less then optimal. To make them excellent candidates for implants and tissue engineering scaffolds, a surface modification is required.

Objectives: Incubating a porcine aortic valve matrix with a platelet gel (PG) concentrate creates a bioactive matrix which is loaded with growth factors. These matrices can be repopulated with mesenchymal stem cells. However, these recellularized matrices still elicit a host immune response. Therefore, the aim of this study was to evaluate the cytotoxicity and cross-linking effect of naturally organic compounds such as quercetin, tannic acid, caffeic acid and catechin on these matrices and to investigate the effect of these cross-linkers on the in vitro growth factor release rate. Materials and methods: Porcine aortic heart valves were decellularized using a patented detergent/enzymatic treatment. Cytotoxicty of the cross-linkers was evaluated by cell culture media supplementation of 10, 100, 1000, 5000, 10000 and 20000µg/mL These concentrations were also used to cross-link the acellular matrices. Mechanical strength of the leaflets was investigated. Also the effect of these cross-linkers on the growth factor release from the PG loaded scaffolds was evaluated by ELISA. Results: Results showed that proliferation of porcine mesenchymal stem cells increased significantly with increasing concentrations of quercetin, tannic acid, caffeic acid and catechin. All compounds except tannic acid significantly increased mechanical strength of the matrices. Moreover, tensile strength of quercetin cross-linked matrices was comparable to the commercially available 0.625% glutaradehyde fixed valves. Furthermore, cross-linking of the matrices resulted in a decreased burst release of growth factors during the first 4 hours but prolonged the release after 24 hours when compared to non-cross-linked matrices. Conclusion: Natural compounds such as quercetin, caffeic acid and catechin can be used to cross-link porcine aortic valve matrices. Moreover, the in vitro release of growth factors can be prolonged which can be very advantageous in the recellularization of these scaffolds.

Growth factors that regulate proliferation, migration, and invasion of ovine mesenchymal stem cells (oMSCs) are not well defined. In this study, we have evaluated five growth factors for their ability to initiate and support in vitro proliferation, migration, and invasion of oMSCs. oMSCs were exposed to different doses and combinations of the growth factors: basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), insulin growth factor-I (IGF-I), connective tissue growth factor, and platelet-derived growth factor-AB (PDGF-AB). Cellular proliferation, motility, and invasiveness were assayed. The most proliferative stimulating growth factors are PDGF-AB+TGF-β and PDGF-AB+IGF-I. Combinations EGF+bFGF and EGF+bFGF+PDGF-AB demonstrated the greatest ability to stimulate migration. Moreover, the triple cocktail EGF+bFGF+TGF-β has the most significant effect on invasion. Different growth factor cocktails are required to enhance proliferation, migration, and invasion. These results may be useful for the development of a tissue-engineered heart valve by stimulating cellular repopulation.

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Electrospinning (ES) of polymer solutions generates non-woven webs of nanofibres. The fibre diameter ranges between 10 nm and 1 μm depending on the operating conditions. Surface functionalisation can be performed by the use of suitable additives. Detailed characterisation of the molecular composition at the fibre surface is a key issue. Biodegradable nanowebs with potential antibacterial activity have been prepared by ES of solutions containing polycaprolactone (PCL) and a functionalising additive with PCL segments and hexyldimethylammonium groups (PCLhexaq). Static secondary ion mass spectrometry with Bi3+ projectiles has been applied to individual nanofibres. The positive ion mass spectra contain several signals with high structural specificity allowing the presence of PCLhexaq to be traced back in spite of its low concentration (0.161.4% w/w relative to PCL) and its structural similarity to the PCL fibre matrix. Imaging of structural ions visualises the homogeneous distribution of PCLhexaq over the fibre surface. Quantifying the surface concentration of PCLhexaq relative to that of PCL reveals electric field-driven surface enrichment of the additive during ES. Finally, nanofibres subjected to leaching in water for up to 72 h have been analysed. The PCLhexaq surface concentration decreases almost linearly with time at a rate of 0.6% h−1.

Nonthermal plasma activation of ultrahigh molecular weight polyethylene (UHMWPE) is performed in different discharge atmospheres (helium, nitrogen, argon, and air) using a parallel-plate dielectric barrier discharge reactor at medium pressure. Obtained samples are characterized via water contact angle, x-ray photoelectron spectroscopy (XPS), and atomic force microscopy. Plasma treatments were found to significantly alter the samples' surface chemical and physical characteristics. Before conducting a biocompatibility study, the influence of postplasma sterilization processes (ethanol rinsing/UV exposure) on the plasma treatment effects were studied. XPS studies showed that samples' surface chemical composition is least affected by UV exposure, thus making this process the most adequate for sterilization. In-vitro evaluation of biocompatibility was carried out by studying human foreskin fibroblast interaction with untreated and plasma treated surfaces. Cell attachment and viability on all plasma treated UHM...

A reduced mortality due to hyperglycaemia was noted since the development of insulin treatment for type I diabetes and various oral hypoglycaemic agents for type II diabetes. Nevertheless the chronic metabolic disorder, Diabetes Mellitus, remains an important cause of morbidity and mortality due to a series of common secondary metabolic complications. Patients with diabetes have an increased tendency to develop infections of the skin. Healing of skin lesions in diabetics evolves often relatively slow and the lesions tend to be more severe than in non-diabetics. Endeavouring to accelerate the healing process of skin lesions in diabetic patients, this preliminary in vitro study investigates the efficacy of green Light Emitting Diode (LED) irradiation on fibroblast proliferation of cells in hyperglycaemic circumstances. In an attempt to imitate the diabetic environment, embryonic chicken fibroblasts were cultured in hyperglycaemic medium (30.000mg Glucose per litre Hanks Medium). LED irradiation was performed three consecutive days with a wavelength of 540 nm and a power output of 10 mW, at 0,6 cm distance from the fibroblasts. Each treatment lasted 3 minutes, resulting in a surface energy density of 0,2 J/cm2. Statistical analysis revealed that LED irradiation at the applied parameters induced a higher rate of proliferation in hyperglycaemic circumstances after irradiation than in the same circumstances without irradiation. Regarding these results the effectiveness of green LED irradiation on cells in hyperglycaemic circumstances is proven. To ensure the effectiveness and to evaluate the value of LED irradiation in vivo , further research is required.© (2004) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The survival rate of replanted and autotransplanted teeth is mainly affected by the reaction of the pulp. Pulpal necrosis can cause periapical inflammation and inflammatory root resorption. The purpose of this study is to learn more about the pulpal changes in autotransplanted immature teeth whose pulp tissue was removed before transplantation. The experimental material consisted of 16 single-rooted teeth with open apices, from a beagle dog (3 months of age). At day 0, 4 teeth were extracted, the pulpal tissues were removed, and the teeth were then transplanted to their contralateral side. The same procedure was carried out on days 9, 16, and 23, each time for 4 single-rooted teeth. Longitudinal paraffin sections were made for histologic investigation. The results showed that, after 7 days, 2 of the 4 teeth had an ingrowth of new tissue over one fourth of their length. After 14 days, all 4 teeth had ingrowth (> or =one fourth of the pulp chamber). At the 21-day observation, more than half of the pulp chambers of all teeth were filled, and, after 30 days, there was total ingrowth in 3 of the 4 teeth. This new tissue consisted of well-organized and well-vascularized connective tissue.

The use of human embryonic stem cells (hESC) in both research and therapeutic applications requires relatively large homogeneous populations of differentiated cells. The differentiation of three hESC lines into highly homogeneous populations of osteoprogenitor-like (hESC-OPL) cells is reported here. These cells could be expanded in a defined culture system for more than 18 passages, and showed a fibroblast-like morphology and a normal stable karyotype. The cells were strongly positive for the same antigenic markers as mesenchymal stem cells but negative for markers of haematopoetic stem cells. The hESC-OPL cells were able to differentiate into the osteogenic, but not into the chondrogenic or adipogenic, lineage and were positive for markers of early stages of osteogenic differentiation. When cultured in the presence of osteogenic supplements, the cells indicated the capacity to achieve, under inductive conditions, a mature osteoblast phenotype. The differentiation protocol is based on a monolayer approach, and does not require any exogenous factors other than fetal calf serum, or co-culture systems of animal or human origin. This method is likely to be amenable to large-scale production of homogeneous osteoprogenitor-like cells and thus overcomes one of the major problems of differentiation of hESC, with important relevance for further cell therapy studies.

An electronic device was fabricated consisting of 2 flexible electronic circuit islands, interconnected by a 7 cm long elastic interconnection, which could be elongated for at least 50%. This interconnection was based on gold conductor tracks following a 2-D spring pattern, embedded in a biocompatible silicone elastomer. The complete device was embedded in the same silicone elastomer. An in vitro cytotoxicity extraction test, executed on small test-samples in accordance with the ISO 10 993-1 guidelines, revealed that the applied silicone encapsulation to these samples functioned as a good seal for at least 8 days.

In the frame of the Flemish Community funded project Bioflex we developed and fabricated an implant for short term (< 7 days) bladder pressure monitoring, and diagnosis of incontinence. This implant is soft and flexible to prevent damaging the bladder’s inner wall. It contains a standard flexible electronic circuit connected to a battery, which are embedded in surface treated silicone to enhance the biocompatibility and prevent salt deposition. This article describes the fabrication of the pill and the results of preliminary cytotoxicity tests. The electronic design and its tests, implantation and the result of the in-vivo experimentation will be presented in other articles.